Faculty Bibliography

It is known that arachidonic acid, in addition to promoting release of dopamine, can inhibit its transport. The present study provides preliminary information on structure-activity relationships for uptake inhibition by rotating disk voltammetry in human embryonic kidney-293 cells expressing the human dopamine transporter. Except for anandamide, all other fatty acids studied at a pretreatment concentration of 80 microM caused significant reductions in Vmax but not Km. Increasing saturation of the hydrocarbon tails (partial saturation: oleic acid, linoleic acid; full saturation: arachidic acid, stearic acid, stearic acid ethyl ester) removed inhibitory activity incrementally, suggesting a role for cis-unsaturation (folding/bending of hydrocarbon tails). The relative lack of effect of 5,8,11,14-eicosatetraynoic acid also supports the idea that less linear structures are less inhibitory on dopamine uptake. Esterification of the free carboxylic group (arachidonic acid ethyl ester) prevented most of the inhibitory activity, arguing against mere membrane lipid disruption. Finally, the endogenous cannabinoid anandamide greatly reduced uptake Vmax accompanied by a small decrease in Km, a potentially important effect on dopaminergic neurotransmission

Although Na+ is crucial for the function of the dopamine (DA) transporter (DAT), its role in the substrate binding step has been questioned. To address this issue, we investigated the effect of Na+ on DA binding by measuring the potency of DA in inhibiting the binding of the cocaine analogue [3H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) in intact cells expressing DAT in their plasma membranes and in membranes isolated from these cells. In cells, Na+ substantially enhanced the potency of DA in inhibiting CFT binding. This effect of Na+ was independent of buffer compositions and substitutes (sucrose vs. NMDG), more pronounced at 4 degrees C than 25 degrees C, and correlated with its stimulatory effect on DA uptake Km. Removing extracellular Na+ had little effect on intracellular concentrations of Na+ and K+, or on membrane potential. These data suggest that extracellular Na+ most likely acts at the transporter level to enhance the binding of external DA during the transport cycle. In contrast, in cell-free membrane preparations the Na+ stimulation was abolished without impairment of the potency of DA in inhibiting CFT binding, regardless of whether sucrose was used to maintain the buffer osmolarity. The difference in Na+ dependence for DA to inhibit CFT binding between plasma membranes of intact cells and isolated membranes raises the possibility that intracellular ion environment, alone or in combination with other cellular factors, plays a critical role in determining DA-DAT interaction and the integration of Na+ modulation in this interaction

To explore structure-activity relationships (SAR) of a novel conformationally constrained lead cis-3,6-disubstituted piperidine derivative derived from (2,2-diphenylethyl)-[1-(4-fluorobenzyl)piperidine-4-ylmethyl]amine (I), a series of compounds was synthesized by derivatizing the exocyclic N-atom at the 3-position of the lead. This study led to the formation of substituted phenyl and heterocyclic derivatives. All novel compounds were tested for their affinity at the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in competing for the binding of [3H]WIN 35 428, [3H]citalopram, and [3H]nisoxetine, respectively. Selected compounds were also evaluated for their activity in inhibiting the uptake of [3H]DA. The SAR results demonstrated that the nature of substitutions on the phenyl ring is important in activity at the DAT with the presence of an electron-withdrawing group having the maximum effect on potency. Replacement of the phenyl ring in the benzyl group by heterocyclic moieties resulted in the development of compounds with moderate activity for the DAT. Two most potent racemic compounds were separated by a diastereoisomeric separation procedure, and differential affinities were observed for the enantiomers. Absolute configuration of the enantiomers was obtained unambiguously by X-ray crystal structural study. One of the enantiomers, compound S,S-(-)-19a, exhibited the highest potency for the DAT (IC50 = 11.3 nM) among all the compounds tested and was as potent as GBR 12909 (1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine). However, the compound (-)-19a was more selective than GBR 12909 in binding to the DAT compared with binding to the SERT and NET. The present results establish the newly developed 3,6-disubstituted piperidine derivatives as a novel template for high-affinity inhibitors of DAT. Structurally these molecules are more constrained compared to our earlier flexible piperidine molecules and, thus, should provide more insights about their bioactive conformations

In our effort to develop high-affinity ligands for the dopamine transporter which might find potential use as cocaine medication, a polar hydroxy substituent was introduced into the piperidine ring of one of our disubstituted lead analogues derived from 1-[2-(diphenylmethoxy)-ethyl]-4-(3-phenylpropyl)piperazine (GBR 12935). Both cis- and trans-3-hydroxy derivatives were synthesized and the racemic trans isomer, (+/-)-5, was further resolved into two enantiomers. Newly synthesized compounds were characterized for their binding affinity at the dopamine, serotonin, and norepinephrine transporter systems in rat brain. The two enantiomers (+)-5 and (-)-5 exhibited marked differential affinities at the dopamine transporter with (+)-5 being 122-fold more potent than (-)-5 in inhibiting radiolabeled cocaine analogue binding (IC(50); 0.46 vs 56.7 nM) and 9-fold more active for inhibiting dopamine uptake (IC(50); 4.05 vs 38.0 nM). Furthermore, the most active (+)-5 was 22-fold more potent at the dopamine transporter compared to the standard GBR 12909. Absolute configuration of one of the enantiomers was determined unambiguously by X-ray structural analysis. In in vivo locomotor activity studies, the enantiomer (+)-5 and the racemic (+/-)-5, but not (-)-5, exhibited stimulant activity with a long duration of effect. All three compounds, (+)-5, (-)-5, and (+/-)-5, within the dose range tested, partially (50%) but incompletely (80%) produced cocaine-like responses in mice trained to discriminate 10 mg/kg ip cocaine from vehicle. Compound (-)-5 was distinctive in this regard in that, unlike (+)-5 and (+/-)-5, it did not affect locomotor activity yet, but similar to them, was able to engender (albeit incompletely) cocaine-like responses

1. Conflicting results have been reported regarding the influence of nitric oxide (NO) and peroxynitrite on dopamine (DA) uptake and release. In the present study, effects of NO donors were studied in rat C6 glioma cells expressing human DA transporter. 2. [(3)H]-DA uptake was inhibited by S-nitroso-thiol S-nitroso-N-acetylpenicillamine, spermine/NO, diethylamine/NO (DEA/NO), (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)-amino]/NO (PAPA/NO), and 3-morphosynodiomine (SIN-1) in a rank order correlating with their half lives as NO donors, whereas no effect was observed for diethylenetriamine/NO and dipropylenetriamine/NO, which release NO very slowly. 3. Hydroxycobalamin, a NO scavenger, but not superoxide dismutase and catalase, enzymes that metabolize superoxide and hydrogen peroxide, respectively, abolished the inhibitory effect of DEA/NO and SIN-1, indicating that they inhibit DA uptake through a mechanism related to the production of NO but unrelated to the formation of peroxynitrite. In consonance, peroxynitrite did not alter DA uptake in the present system. 4. DEA/NO and PAPA/NO reduced [(3)H]-MPP(+) uptake, whereas the release of [(3)H]-MPP(+) was not modified, demonstrating that NO can inhibit uptake of DA transporter substrate without accelerating DA transporter-mediated reverse transport of substrate under the same conditions

Receptor-mediated feedback control plays an important role in dopamine (DA) neurotransmission. Recent evidence suggests that release and uptake, key mechanisms determining brain extracellular levels of the neurotransmitter, are governed by presynaptic autoreceptors. The goal of this study was to investigate whether autoreceptors regulate both mechanisms concurrently. Extracellular DA in the caudate-putamen and nucleus accumbens, evoked by electrical stimulation of the medial forebrain bundle, was monitored in the anesthetized rat by real-time voltammetry. Effects of the D2 antagonist haloperidol (0.5 mg/kg, i.p.) on evoked DA levels were measured to evaluate autoreceptor control mechanisms. Two strategies were used to resolve individual contributions of release and uptake to the robust increases in DA signals observed after acute haloperidol challenge in naive animals: pretreatment with 3beta-(p-chlorophenyl)tropan-2beta-carboxylic acid p-isothiocyanatophenylmethyl ester hydrochloride (RTI-76; 100 nmol, i.c.v.), an irreversible inhibitor of the DA transporter, and kinetic analysis of extracellular DA dynamics. RTI-76 effectively removed the uptake component from recorded signals. In RTI-76-pretreated rats, haloperidol induced only modest increases in DA elicited by low frequencies and had little or no effect at high frequencies. These results suggest that D2 antagonism alters uptake at all frequencies but only release at low frequencies. Kinetic analysis similarly demonstrated that haloperidol decreased V(max) for DA uptake and increased DA release at low (10-30 Hz) but not high (40-60 Hz) stimulus frequencies. We conclude that presynaptic DA autoreceptors concurrently downregulate release and upregulate uptake, and that the mechanisms are also independently controlled during neurotransmission

2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) has been increasingly used as nitric oxide (NO) scavenger. Carboxy-PTIO reacts with NO to form nitric dioxide and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl (carboxy-PTI). In rat C6 glioma cells expressing human dopamine transporter, carboxy-PTIO paradoxically potentiated the inhibition of [(3)H]dopamine uptake by two NO donors, diethylamine/NO and (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)-amino]/NO. Further examinations revealed that carboxy-PTI concentration-dependently reduced dopamine uptake, indicating that the formation of carboxy-PTI may account for the failure of carboxy-PTIO to abolish NO elicited effects. These results suggest that caution should be taken in interpreting data obtained using carboxy-PTIO and probably other NO scavengers

In membrane preparations, CFT, a phenyltropane cocaine analog, and dopamine (DA) interact with the recombinant human dopamine transporter (hDAT) in Na+ -free medium. Na+ markedly increased the transporter's affinity for CFT, but had little or no effect on DA potency for inhibiting CFT binding. Raising [Na+ ] from 20 to 155 mm reduced Li+ -induced increase in DA K (i), but not CFT K (d). The presence of 155 mm Na+ enhanced the tolerance to low pH of CFT Kd but not DA Ki. Leucine substitution for tryptophan 84 (W84L) in transmembrane domain (TM) 1 or asparagine substitution for aspartate 313 (D313N) in TM 6 did not or only modestly enhance the affinity of Na+ -independent CFT binding, and retained the near normal ability of DA, Li+, K+, or H+ to inhibit this binding. However, the mutations significantly enhanced the Na+ stimulation of CFT binding as well as the Na+ antagonism against Li+ and H+ inhibition of CFT binding. In contrast, the mutations neither changed the Na+ -insensitive feature of DA Ki nor enhanced the Na+ protection of DA Ki against Li+ 's inhibitory effect, though they caused Na+ protection of DA Ki against H+ 's inhibitory action. These results are consistent with the existence of binding conformations for DA that are distinguishable from those for CFT, and with a differential association of cation interactions with DA and CFT binding. The mutations likely alter Na+ -bound state(s) of hDAT, preferentially strengthening the positive allosteric coupling between Na+ and CFT binding, and reducing the impact of Li+ or H+ on the CFT binding

The conformation and alignment of cocaine analogues bound to the monoamine transporter proteins were explored using the tensor decomposition 3-D QSAR method. It is proposed from these calculations that the bound conformation of these ligands to the three transporter proteins has the 3beta-aryl substituent in a conformation in which the aryl group is orthogonal or approximately orthogonal to the tropane ring. Based on these results, rigid and semi-rigid tropane analogues were designed, synthesized and their affinities for the monoamine transporters were determined